James M. Harkema

Small-molecule selectin inhibitor protects against liver inflammatory response after ischemia and reperfusion

BACKGROUND The selectin family of adhesion molecules plays a key role in the neutrophil-mediated injury observed after ischemia and reperfusion. In our study, we investigated the effects of TBC-1269, a novel small-molecule, nonoligosaccharide inhibitor of P-, E-, and L-selectin binding, in the liver inflammatory response after 90 minutes of warm ischemia. STUDY DESIGN Total liver ischemia was produced in Sprague-Dawley rats for 90 minutes using an extracorporeal portosystemic shunt. The animals were divided into five groups including: the sham (group 1), ischemic control (group 2) receiving only the vehicle, and the treated groups receiving TBC-1269 at a dose of 25 mg/kg at different times of administration: 15 minutes before reperfusion (group 3), at reperfusion (group 4), and 15 minutes after reperfusion (group 5). The following indices were analyzed: 7-day survival, liver injury tests, liver tissue myeloperoxidase as an index of neutrophil infiltration, and liver histology. RESULTS TBC-1269 treated groups experienced a significant increase in survival compared with controls. Best overall survival, 70%, was observed when TBC-1269 (Texas Biotechnology Corporation, Houston, TX) was administered 15 minutes before reperfusion (p < 0.05). This group also showed a marked decrease (p < 0.05) in liver enzyme levels at 6 hours after reperfusion. Neutrophil migration was also significantly ameliorated (81%), as reflected by decreased myeloperoxidase levels. We observed improved histologic damage scores in the treated group compared with controls (p < 0.05). CONCLUSIONS A small-molecule selectin inhibitor (TBC-1269) had a protective effect in livers subjected to 90 minutes of warm hepatic ischemia and 6 hours of reperfusion by decreasing neutrophil infiltration, migration and subsequent tissue damage. The best protective effect was achieved when the compound was administered 15 minutes before reperfusion. These findings offer a new therapeutic alternative for protection against ischemia and reperfusion injury.

Magnesium-adenosine triphosphate in the treatment of shock, ischemia, and sepsis

PurposeTo review the rationale for adenosine triphosphate-magnesium chloride (ATP-MgCl2) administration in shock, ischemia, and sepsis; the beneficial effects on cellular and organ functions and survival; and possible mechanisms of these effects. Data SourcesCurrent literature review. Study SelectionArticles deemed most pertinent, current, and representative were utilized. Data SynthesisDespite apparent, adequate resuscitation of hypovolemic shock and sepsis in experimental animals and patients, persistent cellular and organ dysfunction is apparent. Disturbances in organ microcirculation and tissue hypoxia appear to play an important role. These disturbances occur when the energy needs are increased. Because of the theoretical benefits of ATP-MgCl2 as an energy source, as well as a vasodilator, the administration of ATP-MgCl2 has been investigated extensively, and considerable evidence suggests that ATP-MgCl2 restores the depressed cell and organ functions following ischemia, hypovolemic shock, and sepsis. ConclusionsATP-MgCl2 improves cellular and organ function and survival following experimental shock, ischemia, and sepsis. Studies also indicate that ATP-MgCl2 can be administered safely in experimental animals and in normal human volunteers, as well as in patients following various adverse circulatory conditions. Further trials should be undertaken to determine the effects on cell and organ function in patients following traumatic shock and sepsis.

Mechanism of immunosuppression following hemorrhage: Defective antigen presentation by macrophages

The mechanism by which simple hemorrhage profoundly impairs the proliferative response of T lymphocytes to mitogen and alloantigen, produces a defect in interleukin-2 generation, and increases the susceptibility to sepsis remains unknown. Since antigen presentation (AP) by the macrophage (M phi) plays a critical role in the antigen-specific activation of T-helper cells and lymphokine production, we investigated whether the function of the M phi as an AP cell is altered following hemorrhage. C3H/HEJ mice were bled to a mean BP of 35 mm Hg, maintained at that level for 1 hr, and then resuscitated. There was no mortality with this model. Control mice were not bled but otherwise treated identically. Immediately after resuscitation the mice were sacrificed and peritoneal M phi (PM phi) as well as splenic adherent cells (SAC) were harvested. AP function was tested by coculturing different numbers of PM phi and SAC with D10.G4.1 cells (2 x 10(4) cells/well) in the presence of conalbumin (300 micrograms/ml). This T-helper cell clone proliferates upon recognition of conalbumin in the context of Iak (a M phi surface membrane glycoprotein), thus directly reflecting M phi AP capability. After 72 hr of incubation, the cultures were pulsed with [3H]thymidine and harvested. D10.G4.1 proliferations induced via AP by PM phi and SAC from hemorrhaged-resuscitated mice were 29 and 24% of control, respectively (P less than 0.05). Thus, we conclude that AP by M phi following hemorrhage is defective despite adequate resuscitation, a mechanism which could explain the state of immunosuppression and enhanced susceptibility to sepsis.

Severe depression of gut absorptive capacity in patients following trauma or sepsis

Although clinical studies suggest enteral, as opposed to parenteral, feeding lowers morbidity and mortality rates following severe trauma and after sepsis, it is unknown whether gut absorptive capacity (GAC) is indeed maintained under such conditions. To study this, GAC was determined in patients with blunt trauma (n = 8) and with sepsis (n = 11) by the 1-hour D-xylose absorption test. Excluded were patients with ileus, nasogastric output of more than 600 mL/24 hours, or residual gastric content of more than 25 mL after the D-xylose test. Trauma patients (ISS 8-14) and patients with intra-abdominal sepsis had an initial D-xylose test within 24 to 48 hours of admission, at 72 to 96 hours, and then weekly until D-xylose absorption had returned to normal. D-xylose (25 g in 200 mL water) was given via nasogastric tube to patients and orally to healthy volunteers (controls: n = 8). Results show that GAC was depressed at 24 to 96 hours in both groups but returned to normal by 1 to 3 weeks after trauma or resolution of sepsis. Thus (1) gut absorptive capacity was severely depressed early after trauma and after the onset of sepsis; and (2) the 1-hour D-xylose absorption test provided a simple, quantitative assessment of GAC in critically ill patients. Hence, therapeutic agents that restore gut absorptive capacity may be useful for further reducing morbidity and mortality rates following trauma or the onset of sepsis.

Pharmacologic agents in the treatment of ischemia, hemorrhagic shock, and sepsis

Abstract The systemic inflammatory response to perfusion deficits, injury, and sepsis is characterized by a myriad of physiologic changes that appear to be mediated by numerous factors. It is not possible to implicate a single factor that is responsible for the pathologic process that leads to multiple organ failure. However, it is apparent that interruption of the systemic-inflammatory cascade at various points can attenuate and, in certain instances, ablate the damage to cells and organs and improve survival. At present, traditional therapy is directed at the effects of this pathologic process. The agents reviewed in this article have the potential to ameliorate or prevent cellular and organ dysfunction. Since many of the mediators have recognizable beneficial effects, the major challenge to any therapeutic intervention will be to allow the continuation of normal inflammation, which is critical for host defense, while limiting the pathologic process, which causes cellular and organ injury. With our increasing knowledge of molecular biology and cellular function in various organs during and following injury, the more directive and specific the therapy. One can envision multimodality therapy that will upregulate certain processes while others are attenuated. At present, clinical trials of ATP-MgCl 2 , pentoxifylline, calcium channel blockers, chloroquine, and nutritional immunomodulation seem indicated. Since tumor necrosis factor, interleukin-1, and interleukin-6 seem to be early mediators of this response, well-controlled and designed studies that modify their action should be undertaken in multi-institutional settings. In this regard, preliminary trials using antibodies to tumor necrosis factor and interleukin-1 receptor antagonists are ongoing.

Cellular dysfunction in sepsis

Abstract Sepsis produces a characteristic hypermetabolic and hyperdynamic circulatory state. If this response is not reversed, multiple organ failure follows with a high mortality rate. The cellular dysfunction that results in overt organ failure is not clear. Changes in oxygen delivery and consumption in septic patients have raised questions concerning the adequacy of cellular energy stores to maintain cellular function and integrity. Animal septic models have, however, allowed direct determinations of adenosine triphosphate (ATP) and mitochondrial function and correlation with other measurements of cell function. In fact, studies indicate that cellular energy stores and mitochondrial function are well-maintained at a time when cellular abnormality can be demonstrated. In early sepsis, is, 10 hours following cecal ligation and puncture, abnormalities in cell membrane function and membrane potential are present, while ATP levels are maintained. In addition, although there is an absolute increase in the rate of gluconeogenesis, the response to substrate and hormonal stimulation in isolated perfused liver from rats in early sepsis is altered. In late sepsis, is, 16 hours after cecal ligation and puncture, ATP levels decrease and lactate increases. These findings are consistent with tissue hypoxia resulting from hypoperfusion in late sepsis. Thus, it appears that cellular functions are adversely affected in early sepsis, suggesting that the persistence of deleterious effects of sepsis, if not reversed, may lead to organ failure. Certainly, when hypoperfusion occurs in this setting, cellular energy stores appear to decrease, increasing the risk of organ failure and death.

L-Selectin Blockade and Liver Function in Rats After Uncontrolled Hemorrhagic Shock

Hemorrhagic shock (HS) and resuscitation can be seen as a global body ischemia-reperfusion (I/R) injury characterized by neutrophil infiltration and organ damage. Liver dysfunction occurs early after HS. Adhesion molecules are needed for the first steps ofneutrophil migration. Thus, the purpose of this study was to investigate the role of L-selectin in the liver after uncontrolled HS and resuscitation. Forty-eight Sprague Dawley rats were subjected to uncontrolled HS and resuscitation. Animals were divided into three groups: sham, uncontrolled HS and resuscitation, and uncontrolled HS and resuscitation with anti-L-selectin treatment. At 6 we evaluated liver injury tests, liver tissue myeloperoxidase (MPO), and liver histology. Survival was followed for 3 days and compared between groups. Statistical analysis included Fishers exact test and one-way analysis of variance. Survival significantly increased from 30% in the control group to 60% in the treated group (p < .05). Hepatocellular and structural injury as well as neutrophil infiltration was significantly decreased in treated animals (p < .05). Thus, blockade of L-selectin resulted in decreased hepatocellular injury and increased survival in our model of uncontrolled HS. Selectins may be important therapeutic targets for blockade in the treatment of HS.Hemorrhagic shock (HS) and resuscitation can be seen as a global body ischemia-reperfusion (I/R) injury characterized by neutrophil infiltration and organ damage. Liver dysfunction occurs early after HS. Adhesion molecules are needed for the first steps of neutrophil migration. Thus, the purpose of this study was to investigate the role of -selectin in the liver after uncontrolled HS and resuscitation. Forty-eight L Sprague Dawley rats were subjected to uncontrolled HS and resuscitation. Animals were divided into three groups: sham, uncontrolled HS and resuscitation, and uncontrolled HS and resuscitation with anti--selectin L treatment. At 6 we evaluated liver injury tests, liver tissue myeloperoxidase (MPO), and liver histology. Survival was followed for 3 days and compared between groups. Statistical analysis included Fishers exact test and one-way analysis of variance. Survival significantly increased from 30% in the control group to 60% in the treated group (p < .05). Hepatocellular and structural injury as well as neutrophil infiltration was significantly decreased in treated animals (p < .05). Thus, blockade of -selectin resulted in decreased hepatocellular injury and increased surL vival in our model of uncontrolled HS. Selectins may be important therapeutic targets for blockade in the treatment of HS.

Organ Protection with ATP-Magnesium Chloride

The heart and other organs are dependent on aerobic metabolism. When the arterial blood flow is reduced to the point that insufficient oxygen is available for oxidative phosphorylation, the production of adenosine triphosphate (ATP) is decreased. This reduction in ATP occurs at a time when more energy is needed to protect the cell against injury. Thus, since ATP plays an essential role in numerous cellular functions, it is not surprising that ischemia and hypovolemic shock produce alterations in cellular and subcellular function in the heart and other organs (1,2). Indeed, the resynthesis of ATP is a major rate-limiting factor following hypovolemic shock and ischemia (3,4). Very low levels of ATP are invariably associated with the irreversible state following myocardial ischemia (5,6). Thus, despite attempts to restore adequate tissue perfusion, including oxygen and substrates, there is a failure to replenish and regenerate ATP during such conditions. This results in persistent and progressive cellular injury that can progress to organ failure. Therefore, replenishing cellular energy following ischemia and hypovolemic shock has been one of a variety of different strategies that have been investigated to prevent or minimize cell injury. Moreover, the most direct approach for raising ATP levels under these conditions appears to be the infusion of ATP rather than the administration of substrates and/or agents that would synthesize it.